UA57079C2 - Pharmaceutical composition of tisoxanid and nitasoxanid (variants) - Google Patents

Abstract

A pharmaceutical composition containing as active agent at least one compound selected among the group consisting of formula (I) and formula (II). The active agent is preferably in the form of particles having a particle size smaller than 200 EMBED Equation.3 and a mean particle size of greater than 10 EMBED Equation.3 . The pharmaceutical compositions are preferably stabilized with at least one pharmaceutically acceptable acid. The pharmaceutical compositions are particularly useful for treatment of opportunistic infections in persons with compromised or suppressed immune systems, and in treatment of infections of trematodes. (I) (II).

Description

Description of the invention

The present invention relates to a pharmaceutical composition containing, as an active agent, at least one 2 compound selected from the group consisting of compounds of the formula (I)

DT on

AND

Ko, 87 UMn-so () and formulas (Iii) - o-so-sn,

H

Kai 8 osmn-so (y

The active ingredient is present mainly in the form of particles having a size of less than 200µm and an average size exceeding 1µm.

The invention also relates to pharmaceutical compositions stabilized by at least one pharmaceutically acceptable salt.

Such pharmaceutical compositions are especially advisable to use in the treatment of infectious diseases caused by opportunistic microorganisms, in persons with impaired or weakened immune systems, as well as in the treatment of infectious diseases caused by trematodes.

There is an urgent need to develop methods of treatment of a number of parasitic and bacterial infectious diseases in people with a compromised immune system (AIDS, cancer patients, elderly and aging patients, patients with transplanted organs, taking immunosuppressive drugs). Another relevant area is infectious diseases caused by trematodes, especially in tropical climates. Thus, there is a need to create a pharmaceutical composition to which people with a compromised immune system could show tolerance and efficacy that would be characterized by stability when stored even in tropical climates. Gee!

More specifically, Tochoriazta dopai is a representative of protozoa and is among the most common causes of latent infection of the central nervous system in the world. Many healthy people are infected with this parasite, but as a rule, the immune system helps the body to cope with this infection. at

T. dopai is the most common opportunistic microorganism in the brain of AIDS patients. At this time, toxoplasmosis is becoming an increasingly large-scale problem not only because of AIDS, but also in

In connection with the wider use of immunosuppressive drugs (for example, those administered to patients with transplanted organs). Toxoplasmosis is usually treated with a combination of pyrimethamine and sulfadiazine. Despite their effectiveness, these medicinal substances do not destroy the protective membranes of the parasite, as a result of which the treatment takes the form of continuous administration of maintenance doses of the medicinal product. The toxicity that occurs with such treatment often leads to discontinuation of the drug, especially in people with a weakened immune system, and relapses. The statistics are also bad: the mortality rate is reported to be about 7095 among patients with immunodeficiency, and the average life expectancy is about four months. "» Cryptosporidiosis is caused by the microscopic parasite Staguriozrogidisht ragmite, which belongs to the simplest protozoa. In people with normal immune functions, diarrhea caused by S. ragmite can be intense and long-lasting, but it is self-limiting. In AIDS patients, cryptosporidial diarrhea is often dangerous for life. It has been established that that 15-2,095 AIDS patients suffer from this very reason. Until now, no consistently effective or generally accepted method of treatment for cryptosporidiosis has been developed. And the pathogen most often found in AIDS patients is Epiegosuo2oop Biepetswi - a microscopic parasite that has been found in almost a quarter It now appears that this tiny parasite may be responsible for a large proportion of the many unexplained cases of malabsorption syndrome, diarrhea, and wasting seen in patients infected with the virus.

Ge) human immunodeficiency. An effective method of treatment in this case is also still unknown.

Patients with a positive reaction to HIV infection may also be affected by some other species of microsporidia, including Epserpaiyyogoop PpePet and sypistia, as well as new species identified as Zeriaia ipiesiipaiiv8.

Recent reports suggest a significant increase in the number of microsporidian infections being spread.

An infectious disease caused by the parasite Isozroga bbeii, clinically indistinguishable from iF) cryptosporidiosis. Being common in countries with a tropical climate, I. reiiii was found in less than 195 cases of patients in the United States, although the actual frequency of the disease may be higher than this percentage.

Rpeshytosuziis sagipi is generally classified as a parasite belonging to the protozoa: the results of some studies indicate that it may be a fungus with which it shares certain genetic sequences. R. sagipi affects, as a rule, the lungs (pneumonia caused by Rpeishptosuvziev sagipi (RSR)).

Successful treatment results are reported in 40-60,956 patients, accompanied, however, by some problems, including drug intoxication, especially in patients with a compromised immune system.

Among the many serious manifestations of an infectious disease caused by the human immunodeficiency virus (HIV), 65 RSD stands out in a special row due to its high prevalence rate, unique distribution among age groups, and high mortality rate RSD is the most serious infectious disease,

caused by opportunistic pathogens, in HIV-infected children: the frequency of RSD diseases among

There are at least 12,905 HIV-infected young children who are not provided with prophylaxis in the first year of life. Many children die quickly immediately after developing RSD.

The complex caused by mycobacterium Amisht (MAS) refers to infectious diseases caused by a family of very similar mycobacterial microorganisms Musobrasiegisht amisht and M. ipigaseiShiageI. When MAS appears in people with an intact immune system, it usually occurs in the form of an infectious disease of the respiratory tract. In patients with AIDS, MAS often disseminates (disseminated MAS or

OMAS), and the disease can spread to almost any organ system. According to the latest 7/0 studies, the bacteria causing MAC were found in 4395 patients who lived for 2 years after the diagnosis of AIDS. There is no standard treatment method for disseminated MAC.

Combinations of appropriate drugs are usually prescribed, and if successful, this treatment should be continued for life. There is an urgent need for a more effective method of treatment.

HIV-infected people are especially susceptible to infectious disease caused by Musobasiegisht (shbregsciov, with /5 the course of the disease accelerates. While the extrapulmonary form of tuberculosis is not characteristic

In HIV-uninfected patients, it is often found in people with a positive reaction to HIV infection. The Centers for Disease Control (US) has issued guidelines for the treatment of tuberculosis that indicate the increasing prevalence of multidrug-resistant tuberculosis (MDR-TB). Mortality among AIDS patients with IOC-TV is very high (approximately 8095), and the disease progresses extremely quickly.

Therefore, there is an urgent need to develop a method of treatment for these infectious diseases, which are so widespread among humans and animals and which threaten their lives.

There is also a need to create a drug with a broad spectrum of action, designed to simplify the process of treating infectious diseases caused by trematodes. At this time, as a rule, it is necessary to first diagnose the pathogenic factor of a specific trematode, and then prescribe a course of treatment with drugs selected specifically for this trematode. Many less developed countries do not have special equipment for i) diagnosing a specific trematode. The development of a drug with a wide spectrum of action would eliminate the need for such a diagnosis.

Zspiziozota tapzopi - schistosoma - is the etiological factor of schistosomiasis - the second (after malaria) most Ge! from a serious tropical parasitic disease of man and the most serious infectious disease of man caused by trematodes. Zspiviozota Ppaetaiobrisht is another important species that is the cause of human infection. More than 200 million people worldwide suffer from schistosomiasis, including several hundred thousand in the United States.

Eazsioia Nerais - the common liver fluke - is primarily the cause of the disease in sheep, but humans are in. optional host. This parasite manages to survive in the presence of a strong immune response of the host. Bithionol has been proposed for treatment, but has not been approved for use in the United States.

Thus, there is a need to create a pharmaceutical composition that would have stability during storage even in tropical climates and a wide spectrum of action against trematodes. "

When conducting research on animals and clinical research in the treatment of humans, it was established that the effectiveness of treatment using compounds of formulas (I) and (I) depends on the size of the particles of the active medicinal substance and the stability of the compounds. ;" It is advisable to use the described pharmaceutical compositions for the treatment of trematode infectious diseases of humans and animals caused by Zspizviovot, for example, Zspivzota tapzvopi, Zspivuzota

Naetayobrisht, Zspivozota tekKopdi, Zspivozota iaropisit, Zspivozota ipiegsaiaisht; Razsioia, for example, with Razsioia Peraiisa and Razsioia didapiisa, Razsioiorvziv bBivzKi; and Oisgosoeitst depagyisit, Ne(egorpuez

Paigegorpuez and Me(adoptive uokodam.

Sh- These pharmaceutical compositions are also effective for the treatment of infectious diseases in people with a compromised immune system, caused by opportunistic microorganisms, for example, Stguriozrogidisht ragmite, 5o 8ozroga breiYi, Epiegosuigoop bBiepeizi, Epiegosuigoop ipiezNpaiiv, Musorasiegit (Shregstioziv, Musobasiegint

Sh-amisht ipigaseiIshiage, Rpesthosuzviiz sagipii and Tokhoriazta dopaii.

The pharmaceutical composition can be prepared in a form convenient for oral administration, for example, in the form of a solid dosage form, a liquid suspension or a paste.

For a more complete understanding of the essence and purposes of this invention, reference should be made to the detailed description below, which is accompanied by drawings that show: Fig. 1 - the percentage of inhibition and viability of the host cell of nitazoxanide in relation to E. ipieziipaiiv; (F) Fig. 2 - percentage of inhibition and viability of the host cell of nitazoxanide in relation to U. sogpeae; ka fig. 3 - the percentage of inhibition and the viability of the cell-host of albendazole in relation to E. ipieusiipaiiiv; Fig. 4 - percentage of inhibition and viability of the host cell of albendazole in relation to U. sogpeae; in Fig. 5 and 6 - a graph of the dependence of the optical density values obtained for each well of the T. dopaii culture on the concentration of the medicinal substance in the culture; Fig. 7 - a diagram based on the analysis of the effectiveness of nitazoxanide in relation to mycobacteria grown in a liquid nutrient medium; Fig. 8 - the percentage of active particles with a size of less than 0 μm. 65 The method of treatment of infectious diseases proposed in accordance with this invention includes the introduction of a pharmaceutical composition containing as an active agent at least one compound selected from the group consisting of desacetyl-nitazoxanide of the formula (1):

H

Mo, Z Mn-so 0) and nitazoxanide of the formula (I): d o-So-sNa

H

Mo MN-so (y

Nitazoxanide (MT7) - the compound of formula (I) - is a non-proprietary name for 2-(acetolyloxy)-M-(5-nitro-2-thiazole)benzamide - a compound first synthesized by Roscinol (Kozzidpoi) and Cavier (Samieg) in 1975 2 mg of nitazoxanide can be dissolved in one ml of dimethyl sulfoxide (DMSO). Nitazoxanide is easily absorbed when taken orally.

Until now, there have been no data indicating that the compounds of formula (I) and/or (II) could be widely and effectively used in the treatment of infectious diseases caused by trematodes, or would be sufficiently non-toxic with the fact that to be transmitted by people even with impaired immunity.

Methods of obtaining and some areas of application of nitazoxanide are disclosed in the US patent Mo3,950,351, as well as in the publications of the author of this invention. Desacetyl-nitazoxanide - a compound of formula (I) - is sometimes called tizoxanide or 4-MT7 and is a metabolite of nitazoxanide.

In international publication M/O 95/28393, the author of this invention disclosed a method of obtaining a pure compound of formula (II), as well as a method of using a composition containing a mixture of compounds of formulas (1) and (1).

It was established that solid particles of the compound of formula (I), compound of formula (II) or their mixtures, having a size in the range of 170-520 μm (average particle size "352 μm), have a very limited effectiveness of EM when administered orally to animals or humans. The effectiveness of such particles is inferior to the effectiveness of existing pharmaceutical products and, therefore, unacceptable for regulatory or commercial purposes.

As a result of tests carried out on dogs, it was also established that oral administration of a single dose equal to 5 Ωg per kg of solid particles of the compound of formula (I) and the compound of formula (II) having a size smaller

BbBμm, caused strong adverse reactions in animals. Me

In addition, it was established that in order to ensure effective and toxicologically safe treatment of infectious diseases caused by parasites, bacteria, fungi and viruses in humans and animals, the pharmaceutical composition - either in a solid dosage form or in the form of an aqueous suspension - should contain an effective amount of the active agent in the form of solid particles having a size of less than 200 µm and containing M compound of formula (I) and/or compound of formula (II), while the average size of active solid particles exceeds 32 10 µm. at

The presence of a high content of particles of the active agent with a size of more than 2000 μm, compared to particles with a size of 5-200 μm, significantly reduces the chemotherapeutic activity of these compounds. In a preferred embodiment of the invention, the mass fraction of active solid particles with a size of more than 4,200 µm in the pharmaceutical compositions proposed in accordance with the invention does not exceed 595. In the most preferred embodiment, the pharmaceutical compositions proposed in accordance with the invention do not contain active C solid particles with a size of more than 200 microns. "» The presence of a high content of particles of the active agent with a size of less than bμm, in comparison with " particles with a size of 5-200 μm, can lead to an undesirable effect of the drug on animals or humans. In addition, it was found that particles that have a size of less than bmcm are more quickly absorbed from the gastrointestinal tract into the bloodstream and, therefore, are ineffective against parasites, bacteria, fungi and viruses that, as a rule, live in the gastrointestinal tract of an animal or a person. -I A skilled person would not be able to conclude that the particle size of the compound of formula (I) and the compound of formula (II) could have such a significant effect on the antimicrobial activity in animals and humans. Thus, for example, in the process of research conducted by the author of this invention, such antiparasitic compounds as, for example, -I 20 albendazole, mebendazole, niclosamide, praziquantel and metronidazole, did not reveal any noticeable difference in antiparasitic activity in animals or humans, which would depend on the size of their particles. In addition, a qualified specialist would not be able to come to the conclusion that the particle size of the compound of formula (I) and the compound of formula (II) could have such a negative effect on the ability of animals or humans to tolerate the introduction of the specified active agent. 59 The compound(s) of formula (I) and (I) can be administered either in the form of a solid dosage form or in the form of

HF) of an aqueous suspension, and it is preferable that the pharmaceutical composition contains an effective dose of the active agent in the form of solid particles of the compound of formula (I) and/or (II), having a size of less than 200 μm, and the average size of the indicated active solid particles is greater than 10 μm, as determined by the Socieke Socpieg 5 100 equipment. This equipment uses a laser light beam 60 with a wavelength of 750 nm, which allows the determination of the size of particles with a diameter ranging from 0.4 to 900 μm by light diffraction. Sample measurements are made in water with a small amount of Tpyop X-100, which is used to increase the wettability and deflocculation of the powder.

It is desirable that the average size of the indicated active solid particles is in the range from 10 to 100 μm, preferably from 20 to 500 μm. Examples of preferred compositions are: bo - a composition for which the mass fraction of the specified active solid particles with a size of more than 1О0Ωm,

is less than 1095; - a composition for which the mass fraction of the specified active solid particles with a size of less than 5 Ωm is at least 5095.

It is desirable that the average size of the indicated active solid particles is in the range from 10 to 100 µm, preferably from 20 to B5 µm. According to the preferred embodiment of the invention, the proposed composition has less than 1095 specified active solid particles with a size of less than 5 μm.

The active agent or agents used in the form of a solid dosage form or suspension are mainly a mixture of solid particles of the compounds of formula (I) and formula (II), the size of which is less than 200 μm, 7/0 and the mass fraction of the compound of formula (I) in relation to the total mass of compounds of formula (I) and formula (II) of the specified mixture is in the range from 0.5 to 2095, preferably from 0.5 to 10905.

The invention also relates to the pharmaceutical compositions described above, which preferably contain at least one pharmaceutically acceptable acid. Examples of such acids are: citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, fumaric acid, adipic acid, malic acid and mixtures thereof.

The most preferred is citric acid. The presence of the indicated acid improves the stability of the active agent or agents.

The ratio of the mass of the pharmaceutically acceptable acid to the mass of the specified active solid particles is preferably between 0.01 and 0.5, preferably between 0.03 and 0.2. It is desirable that the amount of acid is sufficient to maintain the pH of the suspension in the range from 2 to 6, preferably from 3 to 5, and most preferably from 3.5 to 4.5.

Preparation techniques and preferred examples of solid and liquid dosage forms of the pharmaceutical composition are disclosed in international publication UMO/95/28393, the disclosure of which is incorporated herein by reference. It is desirable that the compositions contain a wetting agent and, if possible, a starch derivative, for example, similar to those disclosed in US patent No. 5,578,621, the contents of which are incorporated herein by reference for the disclosure of possible wetting agents and starch derivatives. A wetting agent as described in US Pat. i)

Mo5,578,621, serves as a dispersing agent.

Such pharmaceutical compositions, prepared either in the form of solid or liquid dosage forms, or in the form of pastes or ointments, may, if necessary, contain additional active agents, for example, antibiotics, Ge! zo antiviral agents or proton injection inhibitors. Although it is impractical, it is also possible for such pharmaceutical compositions to contain active solid particles of the compound of the formula (I) and/or the compound of the formula (I), the size of which exceeds 200 µm. at

The compositions may contain excipients known per se for the purpose of preparing dosage forms convenient for oral administration. -

In order to ensure a high degree of effectiveness within a wide range of parasites, bacteria, fungi and viruses, it is advisable that the coefficient of distribution of the indicated active solid particles is in the range from 0.8 to 2, preferably from 1.1 to 1.9, and most preferably exceeded 1.5, and the indicated distribution coefficient is calculated according to the following formula:

Evozh (For 1095)/K0Yu or 095)/2) "de ts - EReo", - distribution coefficient at 90905; - Iodine, - the maximum size of that fraction of particles, which corresponds to 9095 specified active solid particles; with - bo5; - the maximum size of that fraction of particles, which corresponds to 1095 specified active solid particles.

According to one of the specific variants of the invention, the particles of the compound of formula (I) and/or (II) are obtained by the methods described above, after which they are subjected to grinding so that the size of the active particles less than 1095 exceeds 100 µm, the size less than 5095 of the specified active particles exceeded 5μm and the size of less than 1095 of the specified active particles was less than 5μm, and the average size of the particles was in the range from 20 to bΩm. Then these active particles undergo granulation using a mixture containing active solid particles and at least one granulating agent. Examples of 5o granulating agent can be: polyvinylpyrrolidone, water, alcohol, sucrose, hydroxyl cellulose and their

Sh- mixtures. During the granulation process, it is advisable to add at least one pharmaceutically acceptable acid.

Ge) The invention also relates to solid dosage forms containing the composition proposed in accordance with this invention, such as tablets, dispersible tablets, coated tablets, matrices, etc. The dosage form proposed according to the invention contains, for example: - solid active particles with a size of less than 200 µm, and less than 1095 of the specified particles have a size of more than 100 µm, less than 5095 of the specified particles have a size of more than 5 µm and less than 1090 of the specified particles have a (F) size of less 5 μm, and the average particle size is between 20 and 50 μm; ka - at least one granulating agent; - at least one wetting agent; 60 - at least one starch derivative; - at least one pharmaceutically acceptable acid, which is added mainly during the granulation process.

Liquid dosage forms, for example, aqueous suspensions proposed according to the invention, contain, for example: - as an active agent, solid particles containing a compound of the formula (I) and/or a compound of the formula (II) and that b5 have a size of less than 200 μm, and less than 1095 of the specified particles have a size of more than 100μm, less than 5090 of the specified particles have a size of more than 5μm and less than 1095 of the specified particles have a size of less than 5μm;

- at least one granulating agent; - at least one wetting agent; - at least one pharmaceutically acceptable acid, and the pH of the suspension is in the range from 2 to 6, preferably from 3 to 5, most preferably from 3.5 to 4.5; - at least one thickening agent, for example, xanthan gum, guar gum, crystalline cellulose, carob gum, carboxymethyl cellulose or their mixture.

Medicinal forms in the form of a paste or ointment, proposed according to the invention and intended for oral administration, contain, for example: 70 - as an active agent, solid particles containing a compound of formula (I) and/or a compound of formula (II) and having the size less than 200μm, and less than 1095 of the specified particles have a size of more than 100μm, less than 5090 of the specified particles have a size of more than 5μm and less than 1095 of the specified particles have a size of less than 5μm; - at least one wetting agent; - at least one pharmaceutically acceptable acid, and the pH of the suspension is in the range from 2 to 6, preferably from 3 to 5, most preferably from 3.5 to 4.5; - at least one thickening agent, for example, xanthan gum, guar gum, crystalline cellulose, carob gum, carboxymethyl cellulose or their mixture.

Medicinal forms in the form of a paste or ointment, proposed according to the invention and intended for local or intravaginal use, contain, for example: - as an active agent, solid particles containing a compound of formula (I) and/or a compound of formula (II) and having the size is less than 200μm, and less than 1095 of the specified particles have a size of more than 100μm, less than 5090 of the specified particles have a size of more than 5μm and less than 1095 of the specified particles have a size of less than 5μm; - at least one wetting agent; - at least one pharmaceutically acceptable acid, and the pH of the suspension is in the range from 2 to 6, preferably from 3 to 5, most preferably from 3.5 to 4.5; - cetyl alcohol and/or derivatives of glycerides and/or propylene glycol; i) - at least one thickening agent, for example, xanthan gum, guar gum, crystalline cellulose, carob gum, carboxymethyl cellulose or their mixture.

The dry pure compound of the formula (I) and the dry pure compound of the formula (II) were subjected to grinding and sorted by Ge! by particle size using a sieve.

After grinding, the particles of compounds of formula (I), compounds of formula (I) and their mixtures had the granulometric composition shown in Fig. 8. Figure 8 shows the percentage of particles smaller than microns.

It follows from this figure that: и- - the mass fraction of particles smaller than approximately 5 μm is less than 10905; у - the mass fraction of particles larger than approximately 7 Ωm is less than 1095; - the average particle size is approximately 4 Ωm; - the particle distribution coefficient is about 1.73, and the specified coefficient is calculated according to the following formula:

Evozh (For 1095)/K0Yu or 095)/2) - s pe . - EReo", - distribution coefficient at 90905; and - Iodine, - the maximum size of that fraction of particles, which corresponds to 9095 specified active solid particles; - b10o5, - the maximum size of that fraction of particles, which corresponds to 1095 specified active solid particles.

Specific examples of such compositions are disclosed in the following tables. 1 i o - 5 o i (F) ime) i active agents ov

Sucrose 20 mg

Coating:

A hot solution of sugar or a film coating that is sprayed on tablets or granules containing 500 mg of the active agent and the suspension was about 4.1. tv 2 se o

F zo cha (av) t " and formula (II) and the compound of formula (I) as active agents h 4 2 s . o s - av | Pharmaceutical compositions proposed in accordance with the invention are compositions that have a wide spectrum of action against parasites, bacteria, fungi and viruses, especially when administered orally. and The efficacy and safety of the pharmaceutical compositions disclosed above are extremely high in both (Che) animals and humans. More specifically, in clinical studies of patients, it was found that the efficacy of the above-described pharmaceutical compositions are much more effective in the treatment of parasitic infectious diseases than the same dosage forms that use an active compound whose particle size is in the range from 170 to 520 μm (average particle size -352 μm), even in cases where more large particles were administered to patients in doses increasing by a factor of three and for longer periods of time. are listed in table 6

Table 6 60 Comparison of the results of clinical studies of patients using compounds of formula (I) and formula (II), having particle sizes ranging from 170 to 520 μm (average -352 μm), with the results obtained when using compounds of formula (I) and formula ( II), having a particle size in the range from 5 to 200 μm (average - ЗАмкм)

Compound of formula (I) (9895) - compound of formula (II) (295)

Particle size 170-520μm Particle size 5-200μm bo Dose-15-B5Omg/kg/day for 3-7 days Dose- 15mg/kg/day for 3 days

Cured/All-PEL, U Cured/All-PEL, U

Parasite 2 Viaviosuviiv pPotipiv 12/27-4495 10/10-10095

Siagaia Iatria 11/47-3096 50/73-6896

Avsagiv Iptbgisoidev / 3/69-4965 144/179-80965

7/A8-1595 Tgpispygiv Igispioga 58/79-7396

For each of the parasites listed in Table 6, the proportional indicators of treatment effectiveness were significantly better in patients treated with active particles with a size of 5-200 μm than in patients treated with active particles with a size of 170-520 μm, and in in each case, the statistical significance was p«0.02 (using the standard X 2-criterion). This was true even though the doses of the active agent with the larger particle size were generally 195 higher and the duration of treatment was often longer than for those patients administered the pharmaceutical compositions of the active agent with the larger particle size less than 200 μm. No serious side effects were observed in either group.

When testing the drug on animals, results similar to those described above with regard to studies of the drug in the treatment of humans were obtained.

In addition, adverse reactions observed in dogs after oral administration of a single dose amount

BOmg/kg of the compound of the formula (I) and the compound of the formula (I) were not observed in the course of extensive studies on animals using the compound of the formula (I) and the compound of the formula (II), the particle size of which was in the range from 5 to 200 µm (average " 1µm), even when the same or higher dose of these compounds was administered daily for 90 days or more. c 29 In addition, the specified compositions were stable (even when exposed to temperatures up to 402 and bBor-nii (In relative humidity for six months or, when using liquid suspensions, when subjected to suspension in water under the same conditions for three months), that , thereby demonstrating that the active ingredients do not degrade and that the compositions retain their effectiveness for a period of time after their preparation that satisfies both medical and commercial requirements. b

The following examples illustrate the effectiveness of the proposed pharmaceutical compositions. -

EXAMPLES

SEMRTOBROKISISHM RAKMYOM Fr

In the course of a preliminary clinical study, ZO patients with CNID diagnosed with cryptosporidial diarrhea were treated with daily oral administration of nitazoxanide at a dosage of 500-200 Ω.

A group of twenty-eight men were subjected to a course of treatment lasting two weeks or more, and 16 of them were tested for therapeutic response after an eight-week course of treatment. In this last group, 12 men had a 5090th or higher degree of weakening of the daily frequency of defecation, and 10 men had a noticeable reduction (destruction) of the parasite present in the stool, and the presence of this microorganism "was absolutely impossible to detect in four men. In six patients, favorable З7З 70 indicators were recorded in relation to both clinical and parasitological aspects of the course of the disease. c Patients who received large daily doses of the drug for longer periods of time had a higher chance of a positive reaction.

An open-label study of nitazoxanide in cryptosporidial diarrhea caused by

AIDS, made it possible to document a decrease in the frequency of defecation among patients who took the 15 mg drug with a daily dose equal to 500, 1000, 1500 or 200Omg. Test participants had an average cell count of 42 cells/mm? (in the range of 0-303 cells/mmU), the average frequency of daily defecation equal to 6.7 on average for 15 months, the oocytes of Sturiozrogidisht ragmites in feces and no other obvious small intestinal pathogens. Treatment with azithromycin or paromomycin was unsuccessful for almost all trial participants. -and 50 After 23 weeks of treatment, 9 of 13 patients showed complete clinical response (one to three s of daily mostly solid bowel movements), and 4 of 13 patients showed practical clinical response (at least 5090 reduction in daily bowel movements or change in stool consistency , as a result of which at least 7,595 stools had a solid form). By the end of the study, 8 of the 11 patients had completely cleared the parasite, while the other three had a significant decrease in oocyte counts. A tendency to improve receptivity was noted when daily doses of 1000 mg and more were administered and at more

HF) to a long course of treatment. Two subjects had a blistering rash on the skin; more than 9095 patients followed the research scheme for more than four weeks.

EXAMPLES

SEMRTOBROKISISHM RAKMYOM bo Data on dosage ip mygo

Nitazoxanide was dissolved in sterile dimethylsulfoxide (DMSO) and tested on monolayers of cells infected with oocytes of the original S. ragmites at concentrations of 100 µg/ml, 1 µg/ml, µg/ml and 0 µg/ml. A second study was conducted, during which nitazoxanide was tested at concentrations of 20, 2, 0.2 and b 0.02 μg/ml. These concentrations were achieved by serial dilutions with Dulbecco's modified needle complete medium (OMEM) to a final DMSO concentration of 0.595. A control medium was also contacted with 0.596 DMSO.

In the experiment, a cell culture of MOVKE502 cells, grown in 7-mm chambers, and Stguriosrogydint ragmites - ZSNI oocytes (5x107 per well) were used, while the experiment was conducted to compare paromomycin (positive control drug) with nitazoxanide (experimental drug). The materials contained immune anti-Sturiozrogidicht raguit sporozoite rabbit serum (0.190) and fluorescein-labeled goat anti-rabbit antibody (1965).

Analysis of toxicity tests: 200 μl of medium containing nitazoxanide at concentrations of 100, 10, 1, and 0.1 μg/mL, and the appropriate control 70 medium, were placed in two wells of a 9b-well plate holding confluent cell monolayers

MOVKE502, and in two wells without monolayers. Incubation of the medicinal substance on monolayers was carried out at 37°C in an atmosphere of 890 CO. After 24 hours (test 1) and after 48 hours (test 2), MTZ (Owen's solution) and RMZ (foal mare serum) were added to each well. in concentrations of 333 µg/mL and 25 µmol, respectively. The plate was again placed in the incubator in a dark place for two hours to continue the incubation. After 75 two hours, 100 µl of each supernatant was transferred to a new titration microplate and read by the EGISA plate reader (designed for solid-phase immunoenzymatic analysis) at a wavelength of 49 Ohm. The results were recorded and analyzed. The percentage of toxicity was calculated by subtracting the average value of the optical density (OD) of the supernatants of the medicinal substance from the average value of the optical density (OD) of the supernatants of the control media (without the drug), dividing by

The value of OSH of the control medium and multiplying by 100. o Shcheeredova shcha - Oshchikarska g of the substance x 100 oschsredovy shcha

Analysis of initial oocytes of C. raguit: 5x104 oocytes of C. raguit were incubated in nitazoxanide (100, 20, 10, 2, 1, 02, 01 and Ga 0.02 μg/ml) at 37"C on confluent monolayers of MOVKE5O2 cells . The level of infection in each well was determined and analyzed by immunofluorescence analysis after 24-48 hours. The percentage of inhibition i9) was calculated by subtracting the average number of parasites per 10 wells in the wells for testing the medicinal substance from the average number of parasites per 10 wells in the control medium (without the medicinal substance ), by dividing by the number of parasites in the control medium and multiplying by 100. Number in the control medium - Number in the tested drugs x 100

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Gee! The effect of nitazoxanide on initial oocytes of S. raguit:

In test 1, the use of nitazoxanide in concentrations of 10, 1, and 0.1 µg/ml led to the following results: in the levels of inhibition of parasitic infection - 94.4, 77.2, and 51.895, respectively, and in the levels of cell toxicity - 65.1, respectively, 8.3 and 19.395. Although almost complete inhibition of parasitic infection occurred at a concentration of TOmkg/ml, a high level of toxicity was evident. At a concentration of nitazoxanide of 1 μg/ml, the inhibition of parasitic infection and cellular toxicity compared to a concentration of paromomycin of 2 mg/ml had more favorable indicators (77.290% inhibition of parasitic infection and 8.39% cell toxicity for nitazoxanide at a concentration of Tmkg/ml compared with 5195-fold inhibition of parasitic infection and 23.896-fold cell toxicity for paromomycin at a concentration of the latter of 2 mg/ml). 65 In trial 2, the drug substance was modified to provide a more efficient dose distribution with minimal toxicity. Thus, the cultures remained viable for 48 hours instead

24 hours, as observed in trial 1. An incubation period of 48 hours resulted in a higher relative level of cell toxicity, as evident from the results of the paromomycin study in both trials. A concentration of 20 µg/ml of nitazoxanide was still excessively toxic after a 4- to 8-day incubation period, although the cell monolayer was still intact.

It is possible that a high degree of toxicity, which should affect cellular function, also affects the development of the parasite and, therefore, the intensity of the parasitic infection. At a concentration of 2μg/ml of nitazoxanide, significant inhibition of parasitic infection was observed with a relatively low level of cellular toxicity.

Further dilutions resulted in a greater degree of inhibition of infection and a lower degree of toxicity. 7/0 At a concentration of the medicinal substance equal to 2 μg/ml, moderate cellular toxicity and 94.9095-th inhibitory effect indicate that for the infectious disease caused by C. ragmite ip miigo, nitazoxanide used at a concentration of 2 μg/ml , surpasses paromomycin at a concentration of the latter equal to 2 mg/ml (which, for example, in this case has a concentration 1000 times higher).

AN EXAMPLE OF AI

SEMRTOBROVKISIUMRAKMIM

Data on dosage and storage of ip migo:

Batches of nitazoxanide and desacetyl-nitazoxanide (MT2 and MT7dev) were tested against the cells of the original oocytes of C. ragmites and sporozoite-infected monolayers with a destroyed membrane in concentrations of 10, 1, 041 and

O.01mcg/ml. Each compound was dissolved in 100906 dimethyl sulfoxide (DMSO) and diluted to the required concentrations with sterile Dulbecco's modified Needle's medium (OMEM),

Each concentration of nitazoxanide and control media contained 0.02595% DMSO as a constant value.

In the experiment, a cell culture of MOVKE502 cells, grown in 7-mm chambers, was used, and as Sturiozrogidisht ragmite: ZSNI oocytes (5x10 per well), the experiment was conducted with the aim of comparing paromomycin (positive control drug) with nitazoxanide (experimental medicinal drug). . Materials included rabbit anti-Sturiosrogidiitis ragmite sporozoite immune serum (0.195) and fluorescein-labeled goat anti-rabbit antibody (1965). and)

Analysis of toxicity tests: 200 μl of medium containing nitazoxanide at the concentrations mentioned above and the appropriate control medium were placed in two wells of a 9b-well plate containing cell monolayers confluent with He! with MOVKE5O5, and in two wells without monolayers. Incubation of the medicinal substance was carried out on monolayers at 37°C in an atmosphere of 896 CO. After 48 hours, MT5 (Owen's solution) and PMZ (foal mare serum) were added to each well in concentrations of 33-3µkg/ml and 25µmol, respectively. The tablet was again placed in an incubator in a dark place for two hours to continue incubation. After two hours, 100 μl of each supernatant was transferred to a new titration microplate and read by an EGISA (solid-phase enzyme-linked immunosorbent assay) plate reader at a wavelength of 49O0nm. The results were recorded and analyzed

The percentage of toxicity was calculated by subtracting the average value of the optical density (OD) of the supernatants of the medicinal substance from the average value of the optical density (OD) of the supernatants of the control media (without the medicinal substance), dividing by the value of the AD of the control medium and multiplying by 100. 100 « 20 sshsredovy scha sh-v s Cytotoxicity indicators were determined within the following limits: 0.595-per toxicity -0.6-2596-per toxicity -1.26-5096-per toxicity -2.51-7596-per toxicity -3 and 76-10090-for toxicity -4. As a standard value, ;" toxicity levels of O or 1 are considered acceptable levels of toxicity. Toxicity levels of 2, C or 4 are considered high levels of toxicity to the cell monolayer.

Analysis of initial oocytes of S. ragut: with 5x104 oocytes of 3. ragmite were subjected to incubation in nitazoxanide in the concentrations mentioned above at 377 (atmosphere of 895 CO") on monolayers of confluent cells MOVKE5O2. Level of infection in each well

Sh- was determined and analyzed by a computer method of immunofluorescence analysis after 48 hours. The percentage of inhibition was calculated by subtracting the average number of parasites per site in the test well with 5% medicinal substance from the average number of parasites per site in the control environment (without drug

Sh- substances), dividing by the number of parasites in the control medium and multiplying by 100. s Number - in the control environment -. environment and - Number. in. 100 of them have been tested

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Concentration - μg/ml; The number of parasites - the average number of parasites per site (12 analyzed sites); Inhibition, o - the percentage of inhibition of parasitic infection; Toxicity" - the percentage of cell toxicity caused by the use of the drug. 70 From the above data, it can be seen that the inhibitory effect of MT7dez was the same as that of MT72 on the example of HER.

Both nitazoxanide and desacetyl-nitazoxanide were equally effective against

Stguriozrogiditst ragmite in parallel tests, showing 98 and 9495-ne inhibition, which is provided by a concentration of 10 and imkg/ml, respectively, for each compound. For nitazoxanide, a concentration equal to 1 µg/ml was the lowest to provide 9095 µg/ml inhibition, while 50905 µg/ml inhibition could be achieved at lower concentrations of nitazoxanide, such as 0.2, 0.1 and 0.02 µg/ml Jr. Under the same experimental conditions, paromomycin, used as a positive control drug, was 2000 times less effective with an inhibitory effect ranging from 51 to 8395 at a concentration equal to 200 Ωkg/ml.

AN EXAMPLE FOR THEM

E. IMTEZTMAGI5VI M. SOKMEA

2KK-13 cells (rabbit kidney cell line) were added to culture 24-well plates at a concentration of 2.6x10? of cells per well (1.0-milliliter medium; ERMI 1640 with I -glutamine (2 mmol) and 595 heat-inactivated fetal bovine serum). The plates were placed in a CO»o-incubator at 377C overnight, during which the wells were confluent (with one doubling, the concentration could be determined equal to the number of B5X105 cells per well).

Microorganisms of Beria |ipieviipaiv (obtained from tissue culture) were introduced into the host cells at i) a 3:1 ratio with an estimated concentration of 15x10U of microorganisms per well. This ratio resulted in approximately 5095 host cells becoming infected.

Medicinal preparations were dissolved in DMSO, water or methanol (depending on the degree of solubility) in order to (3) obtain several batches at concentrations of 1.Omg/ml. The batches were stored at -70"C. The solutions used in the experiments were prepared in a complete medium for tissue cultivation . All solutions were tested in a triple well. and "c)

The medium was replaced every three to four days (for containing freshly diluted medicinal substances).

On the sixth day (after the addition of parasites and medicinal substances), the cells were examined for toxicity. -

Control cells to which drugs were added, but no parasites, were examined for confluency, IS o) cell morphology, and the presence of dead or motile cells. Cells incubated only with parasites were examined to confirm the infectivity of the parasites (ie, the presence of parasitophorous vacuoles).

Cells incubated with parasites and drugs were examined for host cell toxicity and relative numbers of parasitophorous vacuoles (ie, high, medium, or low).

On the tenth day, 100 μl of 1095 sodium dodecyl sulfate (505) (0.590 - s final concentration) was added to the culture wells in order to rupture the membrane of the host cell and release microsporidia. The total number of parasites present in each well was determined by calculating an aliquot sample on a hemocytometer. The results are expressed as a percentage of inhibition (relative to infected cells that were not injected with medicinal substances).

The results are presented in Fig. 1-4. 1 EXAMPLE M -1 TOCHORIASM OF SOMOIA

Nitazoxanide and desacetyl-nitazoxanide were tested against parasites, and more specifically against (a) strain KN Tochoriazta dopaii maintained by periodic passages in mice. Cell cultures - 50 MKO5 fibroplasts (Viot-Megigequih, France), grown in a 9b-well microplate, were inoculated with T. dopai. In each culture well, with the exception of control well 8 (negative control well), 200 µL of freshly collected tachyzoites were added. After the 4-day incubation period, medicinal solutions were added to the cultures.

Nitazoxanide (MT) and desacetyl-nitazoxanide (aDMT7) were tested at concentrations ranging from 8.107 to 4Omg/L. Medicinal substances were first dissolved in DMSO at a concentration of 2 mg/ml, after which

A number of solutions were prepared for the culture medium. The presence of sediment was not observed.

Gee! Drug solutions were added to the culture (8 wells for each solution), after which the culture tablets were placed in an incubator for 72 hours. Cultures were then fixed with cold methanol. The growth rate of T. dopaii was assessed by the method of solid-phase immunoenzymatic analysis using peroxidase-labeled anti-T. dopaiiy rabbit antibodies. Optical density values were recorded for each well. 60 The results are presented by plotting the dependence of the OC values obtained for each culture well on the concentration of the drug in the culture. Statistical analysis consisted of regression analysis with a 9590 confidence interval and determination of a dose-effect curve based on the AUC values obtained for each medicinal product.

One tablet was stained with Sietwa dye to study the cytopathic effect in cultures. 65 Three separate experiments were conducted. In each experiment, two culture plates were used for each compound: 8 replicate wells were used in each culture plate for each drug concentration.

Results:

The same results were obtained in three series of experiments. Graphical representations of the results of one

Of the most characteristic experiments for each medicinal substance, Fig. 5a, 0, c and ba, B, p.

Nitazoxanide (Fig. 5a, B, c):

No inhibitory effect was noted at concentrations ranging from 10 mg/L to 0.Zmg/L. A significant inhibitory effect was noted at concentrations of 20.Bmg/L with complete inhibition of Tochoriazt growth at concentrations of 22.5 mg/L. However, at concentrations of 22.5 mg/L, marked toxicity of the cell monolayer was noted.70 The results of monolayer examination under the microscope showed that MT at a concentration of 1.25 mg/L causes a cytopathic effect on parasite-infected cells with an increase in the number of parasitophorous vacuoles and a decrease in the number of intracellular parasites. According to the results of the regression analysis, the 5095-na inhibitory concentration could be set equal to 1.2 mg/l.

Desacetyl-nitazoxanide (Fig.ba, bc):

Similar results were obtained with desacetyl-nitazoxanide: no inhibitory effect was noted at concentrations ranging from 10 mg/L to 0.Zmg/L, inhibition was noted at concentrations of 20.Bmg/L, and noticeable toxicity was noted at concentrations of 22.5 mg/l. In 5095, the inhibitory concentration could be set equal to 1.2 mg/l.

The obtained results were reproducible in three separate experiments with the evaluation of the inhibitory effect of the drug on cultures repeated for one or another concentration of each drug.

For both MT and aM1T7, marked inhibition of Tochoriazt growth could be observed at concentrations of approximately 1.2 mg/L with a change in the parasitophorous vacuole, but no noticeable change in the parasite itself.

The results indicate that these medicinal preparations are effective in relation to T. dopaia and that a therapeutic SM effect can be expected even if the concentration of the medicinal preparation in serum or tissues is equal to about mg/l.

EXAMPLE MI

MUSOVASTEKIA

It was established that nitazoxanide exhibits antimicrobial activity against microorganisms that cause C) tuberculosis. The table below shows the data of the analysis of the minimum inhibitory concentration of nitazoxanide and tizoxanide for Musobasiegisht ipigaseishShag, carried out by the method of dilution in agar gel. -

These results are based on several experiments, each of which took about three weeks to perform the agar gel dilution method (a) using agar-agar supplied by MiadiergookK. Data obtained using the ATCC standard Musobrasiegita ipigaseisShiag strain and the standard agar gel dilution test indicate that the minimum inhibitory concentration (MIC) of nitazoxanide against Musobassiegia is 2 µg/ml, and the MIC of tizoxanide is 4 µg/ml.

The minimum inhibitory concentrations of nitazoxanide and tizoxanide in relation to M. ipigaseishag ch i Z s:z" MIC were determined within 3 weeks based on the method of dilution in an agar gel using Midaiengook 7H1I1 agar-agar. The standard strain M. ipigaseishag ATSS 13950 was used in the experiment.

Figure 7 is a diagram based on the analysis of the effectiveness of nitazoxanide in relation to g of mycobacteria grown in a liquid nutrient medium. A colorimetric analysis was applied

MT5, which, when using the agar gel counting method, made it possible to determine growth not in 3 weeks, but in 4 hours. The data presented in Fig. 7 show that when nitazoxanide was added 72 hours after the initiation of culture growth, it had an immediate effect on the development of the culture, which continues, compared to its development in the control environment. A dose of nitazoxanide, equal to Zmkg/ml, stops growth for - the next 24 hours, after which growth retardation is observed, which takes place during the next 2 days.

He Dose of 5 Ωkg/ml was completely bacteriostatic during 144 hours of culture existence.

EXAMPLE OF MINE

SE URTOZOZKIVIIUM RAK MOM

5B The effect of nitazoxanide was tested against Sturiozrogidisht ragmites in experimentally infected mice. Nitazoxadin was supplied by Kotakgk I arogaigieev, I.S., Tampa, Sht. Florida, USA. (F; The total human dose (1g per day for 7 days, i.e. 7g) was modified for use in mice according to the Paget-Barnes method (Radei and Vagpez). The human dose was multiplied by 0.0026 for mice (which weighing approximately 20g) in order to obtain the total amount of medicinal substance required for each carrier organism in the morning and in the evening for 7 consecutive days. Each mouse received 2.bmg per day (7000mgx0.0026/7). Doses were administered orally with using a plastic syringe equipped with a needle with a round tip.

Twenty (20) 2-day-old sucking mice were infected by oral administration of 100,000 oocytes

Sguriozorogidipt raguit, obtained from infected babies. Before administration to mice, oocytes were exposed to 65 concentrations using a sugar solution according to the method described by Feuer and Ellis (Raueg" and

Eiv). Rectal smears were obtained from each mouse and subjected to daily examination using a modified Nielsen smear staining method (MiepI-Meeizep) described by Hraczyk et al. (Sgasguk ei aI.). Release of oocytes in feces began two days after oral infection of animals. On the third day after infection of the animals, 10 mice received 1.3 g of nitazoxanide administered in the morning and in the evening for 7 consecutive days, and the other 10 mice were left as a control group to which no treatment was applied. Rectal swabs were obtained daily during each of the 7 days of treatment and during each of the 7 days after discontinuation of treatment. Oocytes were to be suspended in oil, and their counting was carried out under a microscope based on 100 sections.

Results: 70 The results presented in the table below clearly show that nitazoxanide administered at a daily dose of 2.6mg per day for 7 consecutive days was more effective in Sturiozrogidisht compared to the control group of animals. raguitis, which was manifested in a decrease in the number of oocytes in the feces of infected mice. By the end of the third day of treatment, the use of the test drug resulted in a decrease in the number of oocytes excreted in the feces in 6 out of 10 mice treated with 7/5. At the end of the 7-day course of treatment, a complete cessation of oocyte release was noted, and all treated animals had negative results in the examination of feces compared to a control group of untreated mice. This effect maintained its effect for at least 7 days after treatment, as evidenced by the negative results of the test carried out on the third and seventh days after the end of treatment. 150 | 001740 17100106 4 | 301 201 B | 00 1201700 111010 5150 120100 130100 106 | 00 F ZO 6 | 30 1 00 1 40 | 00 150 10012010 from 30 | 001712о1110о111о1оо о о 100120 100 106 | 00 - zv (All over!) 35 180142 | 00717 8о1710011ю1оо ю

Average 35 00801042 0100000 130 10011000

Ephesus ОО | fruit 11111111 1looyu111111117looyu 5

LH - treated group;

Ef-st - effectiveness of treatment - with EXAMPLE MI "with MUSOVASTEK

Nitazoxanide was compared with the antibiotic isoniazid. The comparison scheme involved the use of BCG (VSO) - bacillus Calmette and Guerin (Vassier de SaItene ei Sytsegip) - as a strain of microbacteria. The sensitivity of this strain was the same as that of M. Schwregsciosis, but this strain is more harmless and, thus, did not require a high level of tuberculosis factor. -Mice were injected with a dose of the drug, equal to 4 mg per day per mouse, prepared using 0.2 ml of sunflower oil. Results in mice treated with nitazoxanide were comparable to the isoniazid group. - 50

Fo o yu me 5 ooo ztwoo vo 65 1675000 1800000 117000) 105000 150000) 750

"SFR is a phosphate-buffered saline solution

THEIR EXAMPLE

EAZSIOG A NERATIS A

The effectiveness of oral nitazoxanide and desacetyl-nitazoxanide in relation to Razorioia Perais was investigated.

Mature Razsioia neraiis were extracted from the bile ducts of livers from calves rejected due to fascilosis in

Nagauz Meai RaskKegv, Banky, pcs. Louisiana, USA, 70 Louisiana State Veterinary Medical Diagnostic Laboratory. Trematodes were washed in sterile saline for one hour and transferred to sterile saline or CSF (pH 7.4) for an additional 3 hours. Then the trematodes were left overnight at 37"С in an atmosphere of 595 CO" in sterile KRMI-rabbit serum (volume ratio 50:50) or in sterile KRMI (pH 7.4).

The culture of ip migo (37"С, 596 СО") was brought to the desired state using the method of modification 75 of Ibarra-Jenkins (Iragta and depKkipe) (7. Ragaziepka. 70:655-661, 1984). Under the conditions of sterility, trematodes were washed twice for 2-3 minutes in Hank's balanced salt solution (Napc) (pH 72) and placed separately in the wells of b-well Linbro culture plates (I iprgo) containing 1 Oml aliquots of the indicated solutions of the medicinal substance in the culture medium. The latter consisted of of sterile LMWH-rabbit serum (volume ratio 50:50) with 296 rabbit blood plus 100 ppm penicillin and 100 ppm streptomycin Only those trematodes with normal activity and morphology were used.

Stock solutions of nitazoxanide (MT7) or its metabolite desacetyl-nitazoxanide (O-MT7) supplied

Kotagk, were dissolved in DMSO (200 Ωkg/ml) and diluted in the culture medium using 100-mm volumetric flasks in order to obtain the specified concentrations of drugs (100, 50, 25, 10, 5, 3, μg/ml). Two control trematodes were included in each replicate well: one in culture medium treated with erythrocytes (RBC) and one in culture medium not treated with erythrocytes (RBC).

Trematodes were examined for the efficacy of drug treatment based on data on death, motility disorders, or morphological changes when compared with untreated trematodes using a backlit panel and illuminated magnifying glass (3

ХХ. them

Results:

Experiment 1: (for YUO-MT7) - trematodes treated with 50 and 100 μg were agonized or died within one | "c) hours. Four out of 7 trematodes were agonized when treated with 25 μg, two were active, and after four hours only one weak trematode remained alive. In the 10-microgram group, there was a decrease in the activity of trematodes

C5 was noted at 1, 3 and 4 hours, and at 7 hours all trematodes were agonized or dead. Reduced M activity was observed in some individuals at 24 h in the bmkg and Zmkg-treated groups, with some delayed onset in the Zmkg treatment: all were dead in the 3- and 5-μg treated wells at 50 h except for one flaccid trematode in each group. Some slowing of activity was noted in the µg-treated group after 42-74 hours, and only 3 active and one agonized trematodes remained alive.

After 91 hours: After 115 hours, only one flaccid trematode remained in the 1 microgram group. The death of three trematodes in the control group containing erythrocytes (RBC) was observed after 66 hours (one trematode), 91 hours (one trematode) and 115 hours (four trematodes). In the control group containing no erythrocytes (RBCs), all trematodes remained alive after 91 hours, and only one died after 115 hours.

Experiment 2: (For MT2) - in comparison with the results obtained for 0O-MT2, a slightly greater activity was noted, which was manifested in an earlier effect on the motility and death of trematodes in 8 replicating plates. o In the 100-, 50-, and 25-microgram groups, all trematodes were dead or agonized after 1 hour except -1 one trematode in the 25-microgram group, which died after 3 hours. A dose-dependent decrease in mobility was observed after 1 hour in each of the other drug-treated groups. In the 10-microgram ("c") group, only one trematode survived after 16 hours. In the 5-microgram group, only 3 trematodes were active -1 50 after b hours, and none were active after 1b hours. After 23 hours, only 2 flaccid trematodes remained alive in the 3-microgram group, which died after 41 hours. One in the 1-microgram group

Me, the trematode died after 16 hours, three after 41 hours and five after 74 hours: Of the trematodes remained active after 91 hours, and one trematode showed activity after 115 hours. In the control group containing erythrocytes (RBC), 7 of 8 trematodes remained alive after 74 hours, C remained alive after 91 hours and two survived after 115 hours. In the RBC-free control group, 6 of 8 trematodes were active at 74 hours, 4 were active at 91 hours, and two remained active at 115 hours. i.e.) The death of trematodes in groups with high dosage (25, 50, 10O0μg) was rapid and was manifested in shriveling and ventral "squeezing". At lower levels of drug treatment, most trematodes by 60 decreased their activity for some time and were more relaxed and "flattened" in a state of agony and death. Starting from the 91st hour, contamination began to affect the results of the experiment in some replicating tablets. For the experiment with Yu-MT7, the excessive development of bacteria and fungi and their associated death in two replicating plates mostly occurred after 115 hours.

For the MT7 experiment, overgrowth and death of trematodes in all replicate plates occurred 65 after 91 hours (in two replicate plates) and after 115 hours (in 5 replicate plates). The results of observations after 139 hours were not taken into account due to the general contamination of most of the tablets.

Conclusions:

The results of the experiments indicate high trematocidal effectiveness of both tested drugs. In relation to R. Nerais - the main metabolite, which is supposed to show activity at the level of the liver - nitazoxanide has slightly greater trematocidal activity than desacetyl-nitazoxanide.

Rapid death of trematodes occurs within 1 hour at a dose of 0-MT7 ip mygo, exceeding 5 Ωkg, within 4 hours at a dose of 25 μg, and within 6-7 hours at a dose of 1 Ωkg. Ten micrograms may be considered a suitable dose for the delivery of the target drug 7/0 substance in a single treatment, if pharmacokinetic data indicate that tissue vital activity levels are maintained for more than 6-8 hours after a single treatment.

High trematocidal activity after 74 hours (three days) was observed for both compounds at dose values equal to C and bBmkg. At a dosage level of 1 μg, a long-term survival of trematodes was observed, which is close to (but not equal to) the indicators of the control group that was not treated with drugs /5 drugs: the delivery of this dose of the medicinal substance to trematodes in liver tissues for 3-4 days can , therefore, have an inadequate therapeutic effect on parasites.

EXAMPLE X

EAZSIOGA SISAMTISA

Nitazoxanide was tested against immature and mature Razsioia didapis in experimentally infected rabbits.

Encysted metacercariae (IMC) of Razcioia didapiisa were collected on a cellophane sheet 28-35 days after infection of snails/snails with Razcioia didapiisa miracidia using the method described by Abdel-Ghani (AraeI-Spapu), according to which the snails were exposed to artificial light daily for ЗОхв in pure dechlorinated tap water. The obtained encysted metacercariae (IMC) were stored under water in a refrigerator at 47C for 5-8 days until they were used to infect experimental animals.

Forty (40) Boskat (Vozsai) rabbits, each of which weighed from 1.5 to 2 kg, divided into two tested groups of 20 animals each, were included in the experiment.

Animals from group 1 were infected by oral administration of 35-40 encysted metacercariae, Ge! from wrapped in a lettuce leaf, which was placed on the root of the tongue of each animal. The rabbits' mouths were kept closed by hand until the encysted metacercariae were swallowed. These animals from group 1 were - used to test the effectiveness of nitazoxanide against immature (4-5 week old) Razsioia didapiisa. at

Animals from group 2 were infected by oral administration of 10-15 encysted metacercariae as described above and used to test the effectiveness of nitazoxanide against trematodes at an early stage of their feeding maturity (more than 10 weeks). yu

Ten animals from group 1 received 35 mg of nitazoxanide in the morning and in the evening for 7 consecutive days 4 weeks after their infection at the immature stage of the parasite in its development cycle. The other ten animals from group 1 were left as a control group in which they were not treated.

Ten animals from group 2 received 35 mg of nitazoxanide in the morning and in the evening for 7 consecutive days 10 weeks after their infection at the stage of maturity of the parasite in its development cycle. The other ten animals from group 2 were kept as a control group in which they were not treated.

All animals received food in the form of a dry ration until the end of the experiment. ;" Seven days after the last dose of nitazoxanide, all rabbits from each group were sacrificed.

The surface of the liver was examined for the presence of dead migratory folds, especially at the immature stage of the parasite in its development cycle. These dead areas were examined using two surgical needles to extract immature migrating trematodes according to the technique described by El-Baha (EI-Wapu). The liver was cut into small pieces, especially around the migrating folds, which were macerated under the microscope with

Sh - for the purpose of extracting the present trematodes. The abdominal cavity and visceral surfaces were washed with warm water. o The water was then collected, filtered and tested for immature trematodes. All collected parasites, as well as their parts, were enumerated in both groups 1 and 2 of treated and untreated animals. ш- Live trematodes had a pink color, were transparent, showed an intact shell, which is easily

He) was extracted from the liver tissue using warm water, while the dead trematodes had a grayish color, were loose and had a torn dead surface. The effectiveness of nitazoxanide was calculated using the following formula:

Effectiveness, 90 av 100 a o where: ko a - the number of trematodes isolated from the feces of animals from the control group;

b - the number of trematodes isolated from the feces of animals from the treatment group. in Results:

The results of the research, as can be seen from Table 7, indicate a significant decrease in the number of immature trematodes isolated from the liver of rabbits from the treatment group, compared to the control group. The average percent reduction in the number of trematodes was determined to be 46.7790 (ranging from 40 to 6096). where

Efficacy of nitazoxanide against immature (4-week-old) R. didapiis in experimentally infected rabbits

At the early stage of parasite maturity, nitazoxanide showed 10090% efficacy (10095% kill), and after examination of the livers of treated rabbits, no pathogen was detected compared to animals from the control group, as shown in Table 8. 11 r enin nyn PI PO CHYN PO po PO UNN volo o 1 i nn nn I U pi FIELD: UNN nyny shi; zv 601f in vvyu11111111111111loo i-yuyu pt PO OLYA POLU by POL: UNN bervdeu////11111101111128011111111106111111111111yu1 yu-

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Nitazoxanide, administered as a daily dose of 70 mg for 7 consecutive days, is moderately effective against Razsioia didapiis in its immature stage and 10095-fold effective against this parasite in its early mature stage.

EXAMPLE XII «

ZSNIZTOZOMA - with Nitazoxanide was tested against Zspiziozota tapzopi and Zspizviozota PpetaibBisht in experimentally infected mice. "» Forty (40) white mice weighing 30 to 50 g were divided into two test groups of 20 animals each. The first group was infected with 300-500 freely moving cercariae of Zspiziozota tapzopi suspended in 0.25 ml of distilled of water and administered to each mouse by intraperitoneal injection. The second group was infected in a similar way, but in this case with cercariae of Zspiziovota Ppetaiiobiit.

After infection, these two groups were in laboratory conditions for a total of 70 days. 7 Seventy days after infection of the animals, ten mice from each group were treated with av! nitazoxanide in the form of a 1.3-milligram dose administered orally in the morning and in the evening for 7 consecutive days. Seven days after the end of the treatment course, all mice were killed, and pathogens were extracted from the liver 7 of each mouse by perfusion using warm (37°C) water. Extracted (Che) schistosomes were enumerated for all animals, both treated and The effectiveness of nitazoxanide was calculated based on the following formula:

Efficiency, 90 av 100 a where: o a - the number of trematodes isolated from the feces of animals from the control group; име) b - the number of trematodes isolated from the feces of animals from the treatment group.

Results: 60 The results shown in Tables 9 and 10 clearly indicate that nitazoxanide administered as a daily dose of 2.bmg for 7 consecutive days was more effective in

Zspiviozota Petaijorisht, where 82.85956-no destruction of pathogens was observed, than in relation to Zspiviozota tapzopi, where the rate of destruction of pathogens reached only 59.9195 compared to the control group of mice. These results are comparable to those obtained by Abaza (Araga) et al. in treating patients when 65 nitazoxanide was not effective against 5. tapzopa as evidenced by positive egg counts after a course of nitazoxanide treatment.

y nick ny shin o yan nn nn I PI yuyu nin nin shi iv ny nn nn I yuyu 00000015

The average is 11111199

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Efficiency ////1111vavv « - p

Claims (44)

The formula of the invention with 1. Pharmaceutical composition for oral administration, containing as an active agent, at least one compound selected from the group consisting of: "sl compounds of the formula (I) - CD on () 8 ЛЖ -0.20 МО, МН--сО (Че) and compounds of the formula (II) Мо-со-сн., 00. Z | And in o mo 5 cMn- syu- 23 2. The composition according to claim 1, according to which the indicated active compound is used in the form of active 60 particles, the size of which is less than 200 μm, and their average size exceeds 10 μm. 3. The composition according to claim 2, according to which the average size of the specified active particles is in the range from 10 to 100 μm. 4. The composition according to claim 2, according to which the average size of the specified active particles is in the range from 20 to 50 μm. b5 5. The composition according to claim 2, according to which less than 1095 of the total mass of the specified active particles have a size exceeding 100 μm. 6. The composition according to claim 2, according to which at least 5095 of the total mass of the specified active particles have a size of less than 50 μm. 7. The composition according to claim 2, according to which less than 1095 of the total mass of the specified active particles have a size of less than 5 μm. 8. The composition according to claim 2, according to which the specified composition contains a mixture of active particles of compounds of formula (I) and formula (II), and the mass fraction of the compound of formula (I) in relation to the mass fraction of compounds of formula (I) and formula (II) of the specified mixture is from 0.5 to 20905. 70 9. The composition according to claim 1, according to which the specified composition additionally contains at least one pharmaceutically acceptable acid. 10. The composition according to claim 9, according to which the specified pharmaceutically acceptable acid is selected from the group consisting of citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, fumaric acid, adipic acid / 5 ACIDS, malic acid and their mixtures. 11. The composition according to claim 9, according to which the specified active agent is used in the form of solid particles having a size of less than 200 μm and an average size exceeding 10 μm, and according to which the ratio of the mass of the pharmaceutically acceptable acid to the mass of the specified solid particles is from 0.01 to 0.5. 12. The composition according to claim 11, according to which the ratio of the mass of the pharmaceutically acceptable acid to the mass of the specified solid particles is from 0.03 to 0.2. 13. The method of treatment of infectious diseases in mammals with a disturbed immune system caused by microorganisms selected from the group consisting of Sguriosrogidit raguit, Isozroga bBeiyi, Epiegosuio2oop Briepeivi, Epserpaiogoop o ipieviipaiiv, Musorasiegisht (shregscioviz, Musobrasiegit amisht ipligaseiIshiage, sch ob Rpeitosuziiv Yisagipii Dopain tochoriast, which includes the introduction of a pharmaceutical composition containing as an active agent at least one compound selected from the group consisting of the compound of the formula (1): i) M on () ra I ve o (2) MO, Mn-so m and compounds of the formula (11): and M Оо-сСО-СНаУ 0. 14. The method according to claim 13, where the specified active agent is in the form of active particles having a size of less than 200 μm and an average size of more than 5 μm. - with 15. The method according to claim 13, where the indicated active agent is in the form of particles with an average size in the range of 20-50 μm. "» 16. The method according to claim 13, where the specified pharmaceutical composition contains at least one pharmaceutically acceptable acid. 17. The method according to claim 16, where the indicated pharmaceutically acceptable acid is selected from the group consisting of 1 citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, fumaric acid, adipic acid , malic acid and their mixtures. (av) 18. The method according to claim 13, where the specified active agent is a compound of formula (1). - 50 19. The method according to claim 13, where the specified active agent is a compound of formula (I). 20. The method according to claim 13, where the indicated mammal is a human and where the indicated active agent is administered in an amount in (Che) range of 5000-2000 mg per day. 21. The method of treatment of parasitic infectious diseases caused by trematodes, which includes the introduction of a pharmaceutical composition containing as an active agent at least one compound selected from the group consisting of the compound of formula (1): o M OH () 6 DK 20 MO Z chn -сСо-- 2 and compounds of formula (11): b5 act 22. The method of treatment of parasitic infectious diseases caused by trematodes, according to claim 21, where the trematodes are selected from the group consisting of Zspiviozota, Ravssioia, RazsioiIorvziv, JuOisgosoeiiSht, Ne(egorPpuez and 70 Meiadopishtv. 23. The method of treating parasitic infectious diseases caused by trematodes, according to claim 21, where the trematodes are selected from the group consisting of Zspiziozota tapzopi, Zspiziozota Ppaetaiiobisht, Zspizviozota tekopdi, Zspiziozota |aropiscit, Zspiziozota ipiegsaiaisht, Razsioia Neraiyisa, Razsigoiosota didapiysa, Ravsioiosota, Oshsioiosorii aepagyisit, Ne(egorpuez Pebegorpuez and Me(adopitiv uoKkodam. 24. The method according to claim 23, where the indicated active agent is in the form of active particles having a size of less than 200 μm and an average size of more than 5 μm. 25. The method according to claim 24, where the indicated active agent is in the form of active particles having an average size in the range of 20-50 μm. 26. The method according to claim 23, where the specified pharmaceutical composition contains at least one pharmaceutically acceptable acid. 27. The method according to claim 26, where the specified pharmaceutically acceptable acid is selected from the group consisting of citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, fumaric acid, adipic acid, malic acid acids and their mixtures. Ha 28. The method according to claim 26, where the ratio of the mass of the pharmaceutically acceptable acid to the mass of the indicated active particles is from 0.010 to 0.5. 29. The method according to claim 23, where the indicated active agent is a compound of formula (1). 30. The method according to claim 23, where the specified active agent is a compound of formula (I). 31. Pharmaceutical paste for local application, containing: He»! as an active agent, a solid particle of at least one compound selected from the group consisting of: compound of formula (I) - Мон (Фор! мнднной МО Мнсо 2 and compound of formula (ІІ) "І) , ryu M o-so-sni with s | D! processing: MO» Mn-so, and the indicated particles have a size of less than 200 μm and an average size exceeding 10 μm; o at least one thickening agent; -1 at least one wetting agent; at least one pharmaceutically acceptable acid, and the pH of the paste is in the range from 2 to 6. (ав) 32. Pharmaceutical paste according to claim 31, according to which said paste additionally contains at least one - 50 additive selected from the group consisting of cetyl alcohol, glyceride derivatives, propylene glycol and their mixtures. 33. A pharmaceutical composition for oral administration containing an active agent subjected to granulation in Me, in the presence of a granulating agent, in which: the specified active agent is used in the form of solid active particles, at least one compound selected from the group consisting of: 22 compounds formula (I) is. M on 9 6 TO 5 -- in MO, МНнсо and compounds of formula (II) b5 and M shk MO"? MH (9:60) and in which the specified active particles have a size of less than 200 μm and an average size exceeding 10 μm. 34. The composition according to claim 33, according to which the specified granulating agent is selected from the group consisting of polyvinylpyrrolidone, water, alcohol, sucrose, hydroxyl cellulose and their mixtures. C5. The composition according to claim 33, according to which the specified granular active solid particles contain at least one pharmaceutically acceptable acid. 36. The composition according to claim 35, according to which the indicated pharmaceutically acceptable acid is selected from the group consisting of citric acid, glutamic acid, succinic acid, ethanesulfonic acid, acetic acid, tartaric acid, ascorbic acid, methanesulfonic acid, fumaric acid, adipic acid 715 acid, malic acid and their mixtures. 37. The composition according to claim 35, according to which the ratio of the mass of the pharmaceutically acceptable acid to the mass of the specified active agent is from 0.01 to 0.5. 38. A pharmaceutical composition for oral administration containing an active agent, a wetting agent and a starch derivative, in which: the specified active agent is used in the form of solid active particles, at least one compound selected from the group consisting of: a compound of formula (I) Мон 9 сч » ИЙ Ж (8) mo b 0 бМсо 2 and compounds of the formula (II) Me M Оо-сСо-СН. 00. - 5 - МО» МНн-со - I c) and in which the indicated active particles have a size of less than 200 μm and an average size exceeding 10 μm. 39. Pharmaceutical composition according to claim 38, which additionally contains at least one pharmaceutically acceptable acid. " 40. A pharmaceutical composition according to claim 38, according to which the active particles are subjected to granulation in the presence of a granulating agent to form a granulated active agent containing the specified active tc compound and a granulating agent, the mass fraction of which is, respectively, from 2 to 99.9795 and from 0 .03 to 1095. h» 41. Pharmaceutical composition according to claim 40, according to which the specified granulating agent is selected from the group consisting of polyvinylpyrrolidone, water, alcohol, sucrose, hydroxyl cellulose and their mixtures. 42. Liquid suspension of an active agent for oral administration, containing: as an active agent, solid particles of at least one compound selected from the group consisting of: 1 compound of the formula (I) - M on Sh) zv -I MO МН- СО -- 2 iChe) and compounds of the formula (II) F) АХ ХК Хо ю MO» мн-со 60 and the specified particles have a size of less than 200 μm and an average size exceeding 10 μm; and at least one pharmaceutically acceptable acid, and the pH of the suspension is in the range from 2 to 6. 43. The suspension according to claim 42, according to which the pH of the suspension is in the range from 3 to 5. 44. The suspension according to claim 42, which additionally contains a granulating agent. b5